Yang B et al. (2026), Ccdc57 regulates cilia and left-right patternin...

XB-ART-61753

Biol Open 2026 Feb 15;152:. doi: 10.1242/bio.062495.

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Ccdc57 regulates cilia and left-right patterning in Xenopus.

Yang B, Mis EK, Zhou X, Aslam F, He J, Lu X, Fan H, Guo T, Deniz E, Luo H, Khokha MK.

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During embryogenesis, the establishment of left-right (LR) asymmetry depends on directional fluid flow generated by motile cilia within the left-right organizer (LRO). Disruption of this process can lead to laterality disorders such as situs inversus, heterotaxy, and congenital heart defects. Here, we identify CCDC57 as a regulator of ciliary function and LR patterning. Depletion of ccdc57 via morpholino oligonucleotides (MOs) led to abnormal cilia in the multiciliated cells of the embryonic epidermis of Xenopus. Additionally, LR markers, dand5 and pitx2c were misexpressed resulting in defects in normal rightward cardiac looping. Finally, we identified a patient with situs inversus carrying compound heterozygous CCDC57 missense variants. We tested these variants in Xenopus depleted of ccdc57. Wild-type human CCDC57 mRNA, but not the patient variants, rescued ciliary structure and function. These findings establish ccdc57 as a regulator of LR patterning and suggest its potential involvement in human laterality disorders.

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???displayArticle.pmcLink??? PMC12969765
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Species referenced: Xenopus tropicalis
Genes referenced: ccdc57 cep164 cep63 dand5 dnah5 dnali1 pitx2 rsph9
GO keywords: heart looping [+]
???displayArticle.antibodies??? Arl13b Ab3 Phalloidin Ab3 Tuba4a Ab15
???displayArticle.morpholinos??? ccdc57 MO1
gRNAs referenced: ccdc57 gRNA1 ccdc57 gRNA2

???displayArticle.disOnts??? ciliopathy [+]

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	Fig. 1. Depletion of ccdc57 leads to edema and cardiac looping defects. (A) Illustration of the ccdc57 targeting strategy using both MO and CRISPR-Cas9. The MO blocks the start codon in the coding region, while two different CRISPRs target exons 11 and 16. (B) Normal tadpole heart looping (D-loop) and the abnormal heart loop (L-loop and A-loop) at stage 42 Xenopus. These are ventral views with anterior to the top and the red outline highlights the heart and outflow tract. For embryos with edema, the internal organs exhibit abnormal morphology, and heart looping cannot be scored. Scale bar: 500 μm. (C) The percentage of edema in Crispr1, Crispr2 and MO injected groups. ‘n’, represents the number of embryos. **P<0.0001 according to Chi-Square test. (D) After selecting out the edema embryos, the percentage of abnormal heart looping in Crispr1, Crispr2 and MO injected groups. ‘n’, represents the number of embryos without edema. **P<0.0001 according to Chi-Square test. [[ Abbreviations: MO = morpholino ]]
	Fig. 2. Depletion of ccdc57 alters global LR patterning. (A) Illustration of relevant stages for this experiment. Embryos were injected at 1-cell stage and then collected at stage 16 or 19 for dand5 and stage 28 for pitx2c. (B) The depletion of ccdc57 leads to pitx2c abnormalities. The leftmost panel (blue outline) shows the normal pitx2c expression in the left mesoderm. The other panels show examples of abnormal pitx2c expression as right sided (red), bilateral (green), or absent (purple). All views are ventral with anterior to the top. The arrow indicates pitx2c expression in lateral mesoderm, scale bar: 200 μm. (C) The graph depicts the percentage of embryos with abnormal pitx2c expression. ‘n’, represents the number of the embryos cumulatively in injection groups **P<0.0001 (Chi-square test or Fisher's exact test). (D) At stage 16 (early), dand5 is expressed bilaterally in the LRO (leftmost box outlined in blue). Examples of abnormal dand5 expression are shown including R<L (green) and R>L (red). These are ventral views of dissected LROs with anterior to the top, scale bar: 100 μm. (E) The percentage of embryos with dand5 expression types at stage 16. ‘n’, represents the number of the embryos. ‘ns’ indicates no significant difference between the MO and UIC group. Fisher's exact test. (F) At stage 19 (post flow), the cilia-driven flow suppresses dand5 signal on the left side (leftmost box with blue outline). Embryos with abnormal expression included, R<L (green) and R=L (red). Scale bar: 100 μm. (G) The percentage of embryos with dand5 expression types at stage 19. ‘n’, represents the number of the embryos. Fisher's exact test. **P<0.0001. [[ Abbreviations: MO = morpholino, UIC = uninjected control ]]
	Fig. 3. Depletion of ccdc57 alters epidermal cilia and LRO area. (A-C) The extracellular fluid flow driven by epidermal cilia is visualized using OCT imaging. Endogenous particles (cellular debris) are present within the intravitelline space, and their movement can be tracked by OCT in vivo. Temporal color coding depicts particle trajectory over time. The color bar represents color versus the corresponding frame number in the color-coded image. Based on the trajectory map over 300 frames, we classified the flow as normal flow (A), slow flow (B), or no flow (C), scale bar: 100 μm. (D) Based on OCT imaging, percentages of embryos with normal, slow, and no-flow in uninjected controls and ccdc57 depleted tadpoles. ‘n’, represents the number of the embryos, Chi-square test, **P<0.0001. (E,F) Cilia are reduced in MCCs when ccdc57 is depleted with MO. Cilia were marked with anti-acetylated-tubulin (green) and actin marked with phalloidin (red). Zoomed-in cilia image is shown in the blue boxed area. Scale bars: 50 μm. (G,H) Cilia in the LRO stained by anti-arl13b (red) and actin via phalloidin (green) to mark the cell borders. Scale bars: 50 μm. (I) Total area of the LRO. t-test. **P<0.0001. (J) Cell number in LRO. t-test. ‘ns’ indicates no significant difference. (K) Cilia number in LRO. t-test. ‘ns’ indicates no significant difference. (L) Number of cilia per cell. t-test. ‘ns’ indicates no significant difference. [[ Abbreviations: MO = morpholino, LRO = left–right organizer ]]
	Fig. 4. Dextrocardia in patient with CCDC57 variants. (A) The pedigree of the individual with CCDC57 variants. The arrow indicates the individual with situs inversus. (B) CT scan of the patient reveals dextrocardia. R, right. (C) Sanger sequencing also demonstrates the two variants of CCDC57 determined by exome sequencing. (D) The multiciliary beat frequency of the patient and healthy controls measured at 37°C. Each data point represents the average ciliary beat frequency within a random area. t-test. ***P<0.001. (E) The domain prediction of the CCDC57 protein and the locations of the identified variants. The Alphafold3 prediction of the CCDC57 protein structure. The superposition of the PDB 3D Viewer reveals that the amino acid changes from the patient variants have an impact on the helical angles of the protein. Additionally, the position of the microtubule-binding domain also displays significant changes in the presence of the patient variants.
	Fig. 5. Patient variants in CCDC57 affect protein function. (A) Illustration of injection scheme. The MO was injected at one cell stage depleting ccdc57 throughout the embryo, and at the 2-cell stage, the human mRNA (with or without patient variants) and GFP-tracer mRNA were injected in one cell and embryos selected with targeting to either the right or left side. (B) Cilia driven epidermal flow is visualized using red microspheres over the period of 20 s. (C,D) Cilia-driven epidermal flow visualized by OCT. Temporal color coding depicts particle trajectory over time. The color bar represents color versus the corresponding frame number in the color-coded image. Based on the trajectory map over 300 frames. (E,F) Anti-acetylated tubulin staining of the two sides of the same embryo, the cilia on the MO only side are reduced compared to the opposite side where the human mRNA is injected. (G) The immunofluorescence staining of anti-acetylated tubulin (green) and phalloidin (red) in both CCDC57-variant mRNA injected embryos. (H) Velocity of particles measured by OCT are calculated on both sides of the embryo and the ratio determined between the sides. Therefore, a ratio of 1 indicates that two sides have similar flow velocities (UIC and MO alone injected). UICs and MO both have a ratio of 1 as the velocity on both sides of the embryo are similar. In the case of the wild-type human mRNA, the mRNA injected side is rescued, and the ratio is greater than one. This is significantly different compared to the same experiment with patient variant mRNAs. t-test. P<0.05. *P<0.01. [[ Abbreviations: MO = morpholino, UIC = uninjected control ]]
	Fig. S1. Using ICE Synthego, we verified that CRISPR/Cas9 edited the proper cut site. Genotyping results to confirm the knockdown of the ccdc57 gene. For Crispr-1, 69% of alleles have an out of frame indel and for Crispr-2, 61% of alleles have an out of frame indel.
	Fig. S2. Immunofluorescence staining of DNAH5, DNALI1, RSPH9, and CEP164 in respiratory multiciliated cells from healthy controls.
	Fig. S3. Immunofluorescence staining of DNAH5, DNALI1, RSPH9, and CEP164 in respiratory multiciliated cells from patient.